EP2885407B1 - Pektinabbauende enzyme aus macrophomina phaseolina und verwendungen davon - Google Patents

Pektinabbauende enzyme aus macrophomina phaseolina und verwendungen davon Download PDF

Info

Publication number
EP2885407B1
EP2885407B1 EP13829902.9A EP13829902A EP2885407B1 EP 2885407 B1 EP2885407 B1 EP 2885407B1 EP 13829902 A EP13829902 A EP 13829902A EP 2885407 B1 EP2885407 B1 EP 2885407B1
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
phaseolina
polypeptide
isolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP13829902.9A
Other languages
English (en)
French (fr)
Other versions
EP2885407A2 (de
EP2885407A4 (de
Inventor
Maqsudul Alam
Mohammed Shahidul ISLAM
Mohammed Mosaddeque HOSSEN
Mohammed Samiul HAQUE
Mohammed Monjurul ALAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bangladesh Jute Research Institute
Original Assignee
Bangladesh Jute Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bangladesh Jute Research Institute filed Critical Bangladesh Jute Research Institute
Priority to PL13829902T priority Critical patent/PL2885407T3/pl
Publication of EP2885407A2 publication Critical patent/EP2885407A2/de
Publication of EP2885407A4 publication Critical patent/EP2885407A4/de
Application granted granted Critical
Publication of EP2885407B1 publication Critical patent/EP2885407B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01011—Pectinesterase (3.1.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01171—Rhamnogalacturonan hydrolase (3.2.1.171), i.e. rhamnogalacturonase
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02002—Pectate lyase (4.2.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02023—Rhamnogalacturonan endolyase (4.2.2.23)

Definitions

  • the present invention relates to isolated polypeptides from M. phaseolina having pectate lyase activity and isolated polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides.
  • the invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.
  • the polypeptides of the invention can be used in, amongst other things, the textile industry for the retting and degumming of fibre and fabrics; in food industry for the extraction of fruit and vegetable juice; in paper industry for the production of good quality paper; and also for fermentation of coffee and tea, oil extraction and pectic waste water treatment.
  • Pectin is a group of complex heteropolymers, present in the middle lamella of the primary plant-cell wall and act as intercellular cement. They also have an important function in the water-regulation of plants due to their colloidal nature.
  • the middle lamellas which are situated between the cell walls are mainly built up from protopectin (an insoluble form of pectin).
  • Pectin is composed of two different definite regions: “smooth” and “hairy” regions.
  • the “smooth” region consists of a backbone of ⁇ -1, 4-linked D-galacturonic acid, forming a polygalactouronic acid with some of the carboxyl groups esterified with methanol ( Rouse A. Pectin: distribution, significance. In: Nagy S, Shaw P, Veldhuis Meds. Citrus Science and Technology, Vol I. Westport CT: AVI publishing Inc. 1977 ).
  • the galacturonic acid backbone is broken down by ⁇ -1, 2-linked L-rhamnose unit.
  • Pectin/pectate lyases depolymerize pectin in smooth region, which cleaves glycosidic bonds via ⁇ -elimination to yield oligomers that are 4, 5-unsaturated non-reducing end ( Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, Henrissat B. A hierarchical classification of polysaccharide lyases for glycogenomics. Biochem J. 2010;432(3):437-444 ).
  • Pectin esterase hydrolyses the pectin to methanol and polygalacturonic acid.
  • This enzyme can be produced by several fungi including Aspergillus sp, Botrytis cinerea, Fusarium monilforme, Rhizopus stolonifer, Trichoderma sp. etc ( Polizeli ML, Jorge JA, Terenzi HF.
  • Aspergillus is the major source for the commercial production of pectin esterase ( Torres EF, Aguilar C, Esquivel JCC, Gonzales GV. Pectinase. In: Enzyme Technology, Pandey, A., Webb, C., Soccol, C.R., Larroche, C (Eds), Asiatech Publishiers Inc., New Delhi, India, 2005. pp. 273-296 ).
  • Pectin-degrading enzymes are important tools in the food industry, primarily for fruit and vegetable processing such as fruit juice production or wine making. Other areas of applications include the pulp and paper industry ( Reid I, Ricard M. Pectinase in papermaking: solving retention problems in mechanical pulps bleached with hydrogen peroxide. Enz. Microbiol. Technol. 2000;26:115-123 ), animal feed ( Barreto de Menezes TJ, Salva JG, Baldini VL, Papini RS, Sales AM. Protein enrichment of citrus wastes by solid substrate fermentation. Proc. Biochem.
  • enzymes are used either individually or in a cocktail (mixed) form in the industry.
  • individual enzyme such as pectin esterase required for gellification
  • combination of different enzymes such as pectin esterase with polygalacturonase required for liquefaction of plant material ( Heldt-Hansen HP, Kofod LV, Budolfsen G, Nielsen PM, Wegtel S, Bladt T. Application of tailor made pectinases. In: Visser J and Voragen AJG (eds), Pectin and Peactnases. Progress in Biotechnology. 1996;14:463-474 ).
  • EP 0 278 355 describes the cloning of the pectin lyase gene, the sequence thereof and the expression.
  • EP 0 353 188 adds some other pectin lyases.
  • EO 0 421 919 discloses two polygalacturonases and another endo-polygalacturonase has been disclosed in EP 0 388 593 . Both of these patent applications used Aspergillus niger as the source of the gene.
  • WO 94/14952 describes three enzymes with endo-polygalacturonase activity which is obtainable from Aspergillus aculeatus.
  • no publication was reported on cloning of genes encoding pectin degrading enzymes from M. phaseolina.
  • the present invention takes a genomics approach to disclose the genes and their encoded proteins of the pectin degrading enzymes derived from M. phaseolina that can be used in, among other things, industrial processes or purposes.
  • the present invention relates to pectin degrading enzymes which are derivable from M. phaseolina.
  • the current disclosure also relates to the use of fungus M. phaseolina in the degradation of pectin.
  • the primary object of the present invention is to disclose the sets of nucleotides sequences encoding pectate lyase (SEQ ID Nos. 1, and 2, of the fungus M. phaseolina.
  • SEQ ID Nos. 1, and 2 For each gene of the invention, an open reading frame (ORF) sequence was derived manually from the respective genomic sequence by deleting predicted intron sequences and splicing together exon sequences.
  • Vectors, expression vectors, and host cells comprising the enzyme genes are also included.
  • the invention provides deduced polypeptide sequences from the ORF sequences of the genes.
  • the polypeptide sequences of the invention correspond to those of pectate lyase (SEQ ID No. 3.
  • the present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
  • Another object of the present invention is to provide the molecular biology and genetic information of the genes and enzymes set forth in the primary object to be exploited/utilized for the regulation, conversion of pectin degradation for the production of valuable industrial products.
  • the object of present invention is to facilitate in vitro production of the pectin degrading polypeptide
  • the present invention also includes an expression construct capable of expressing polypeptide containing at least 80% sequential amino acids as set forth in SEQ ID No. 3.
  • the expression construct has inserted DNA or cDNA with sequential nucleotide as set forth in SEQ ID No. 2.
  • Another object of the present invention discloses a recombinant gene construct comprising a polynucleotide template having nucleotide sequence set forth in SEQ ID No. 2, wherein the polynucleotide template is expressible in a host cell to produce an enzyme which degrade pectin.
  • the recombinant gene construct further comprises a promoter region operably-linked to enhance expression of the polynucleotide template.
  • One of the preferred embodiments of the present invention is the ms6 strain of M. phaseolina isolated from an infected jute plant.
  • the isolated polypeptide is also preferably derived from this strain.
  • the object of the present disclosure is to provide a potential commercially feasible way to isolate pectin degrading enzyme from M. phaseolina in order to keep up with the increasing global demand for, amongst other things, the production of fruit juice, textile products, pulp and paper, coffee, tea and oil extraction.
  • Further object of the disclosure is directed to utilize the pectin degrading substances in processing of animal feeds, fruit juice, textile products, pulp and paper, coffee, tea and oil extraction, retting/degumming of bast fibres.
  • kits may comprise polynucleotides and/or polypeptides corresponding to one or more M. phaseolina genes of the invention, antibodies, and/or other reagents.
  • the invention provides an isolated polynucleotide sequence which is at least 80% identical to the nucleotide sequence set forth in SEQ ID No. 1 or 2, or any combination thereof or the complement thereof, wherein said polynucleotide encodes a pectate lyase.
  • the invention provides an isolated nucleic acid probe that hybridizes under medium stringency conditions to an isolated polynucleotide sequence comprising a nucleotide sequence set forth in SEQ ID No. 1 or 2, or any combination thereof or the complement thereof, wherein said polynucleotide encodes a pectate lyase, and wherein said medium stringency conditions include hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50 to about 65°C.
  • SSC sodium chloride/sodium citrate
  • the invention provides a nucleic molecule comprising the isolated polynucleotide sequence of the first aspect uninterrupted by stop codon within a coding sequence that encodes a heterologous protein or peptide.
  • this invention provides a recombinant vector comprising the isolated polynucleotide sequence of the first aspect.
  • the invention provides an expression construct comprising the isolated polynucleotide sequence of the first aspect, wherein the polynucleotide sequence is operatively associated with a regulatory nucleotide sequence containing transcriptional or translational regulatory signals, or both, that controls expression of the nucleotide sequence in a host cell.
  • a sixth aspect of the invention provides a genetically engineered host cell comprising the isolated polynucleotide sequence of the first aspect.
  • a seventh aspect of the invention provides a method of making polypeptide comprising the steps of:
  • An eighth aspect of the invention provides an isolated polypeptide comprising an amino acid sequence which is at least 80% identical to an amino acid sequence set forth in SEQ ID No. 3 or any combination thereof, and displays the catalytic activity of pectate lyase.
  • a ninth aspect of the invention provides a chimeric protein comprising a polypeptide of the eighth aspect fused via a covalent bond to an amino acid sequence of a second polypeptide.
  • a tenth aspect of the invention provides a host cell, comprising the expression construct of the fifth aspect.
  • an eleventh aspect of the invention provides a transgenic fungi of M. phaseolina, comprising the expression construct of the fifth aspect.
  • the present invention and related disclosure provides the nucleotide sequences of M. phaseolina genes involved in pectin degradation.
  • the genes encode proteins with pectin degrading enzyme activity that is either in use in an industry including food, animal feed, paper, oil extraction, textile, waste water or of interest to an industry. Described herein below are the genes of the invention and other genes of the disclosure, their identification, characterization, modification, and methods of usage in various industrial processes.
  • the nucleotide sequences of M. phaseolina genomic DNA was obtained by a whole-genome random shotgun DNA sequencing effort.
  • the genomic DNA was prepared from an isolate of M. phaseolina ms6 which was isolated from the infected jute ( Corchorus spp .) plant.
  • the generated nucleotide sequences were assembled to form contigs and scaffolds by the Newbler assembler.
  • the nucleotide sequences were initially annotated by software programs, such as Augustus, Glimmer M (The Institute of Genome Research, Rockville, Md.) and Evidence Modeler (EVM), which can identify putative coding regions, introns, and splice junctions. Further automated and manual curation of the nucleotide sequences were performed to refine and establish precise characterization of the coding regions and other gene features.
  • genomic sequences of the invention and the sequences of the disclosure that encode the pectin degrading enzymes are identified primarily by comparison of nucleotide sequences of M. phaseolina genomic DNA and the nucleotide sequences of known enzyme genes of other microorganisms.
  • ORFs Open reading frames
  • gene is defined as the genomic sequences of the fungus M. phaseolina, particularly polynucleotide sequence encoding polypeptide of the series of enzymes.
  • the term can further include nucleic acid molecules comprising upstream, downstream, and/or intron nucleotide sequences.
  • ORF open reading frame
  • a “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, such as an amino acid or polypeptide, when the sequence is expressed.
  • the coding sequence may comprise untranslated sequences (e.g., introns or 5' or 3' untranslated regions) within translated regions, or may lack such intervening untranslated sequences (e.g., as in cDNA).
  • a "polynucleotide” is a nucleotide sequence such as a nucleic acid fragment.
  • a polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • An isolated polynucleotide of the present invention may also be derived from SEQ ID No. 1, or the complement of such sequence.
  • An isolated polynucleotide of disclosure may also be derived from SEQ ID No s, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59 and 62, or the complement of such sequences.
  • Isolated means altered “by the hand of man” from the natural state. If a composition or substance occurs in nature, it has been “isolated” if it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living plant or animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
  • recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial or mammalian) expression systems.
  • Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal expression systems. Polypeptides or proteins expressed in most bacterial systems, e.g., Escherichia coli, will be free of glycosylation modifications; polypeptides or proteins expressed in fungi will be glycosylated.
  • vector refers to a plasmid, phage, cosmid, yeast, or virus, an artificial replicating sequence (ARS) or an artificial chromosome for expressing a polypeptide from a nucleotide sequence.
  • vector is also intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector is another type of vector, where additional DNA segments may be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • expression vector is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention, which is operably linked to additional nucleotides that provide for its expression.
  • expression construct can comprise an assembly of a genetic element(s) having a regulatory role in gene expression, for example, promoters or enhancers, or a coding sequence which is transcribed into RNA, mRNA and translated into protein, and which is operably linked to promoter or appropriate transcription initiation and termination sequences.
  • operably linked denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
  • host cell includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • recombinant host cells generally means cultured cells which comprises a recombinant transcriptional unit, and will express heterologous polypeptides or proteins, and RNA encoded by the DNA segment or synthetic gene in the recombinant transcriptional unit.
  • the cells can be prokaryotic or eukaryotic.
  • Polypeptide as used herein, is a single linear chain of amino acids bonded together by peptide bonds, and having a sequence greater than 100 amino acids in length.
  • promoter refers to a nucleic acid sequence that functions to direct transcription of a downstream gene.
  • the promoter will generally be appropriate to the host cell in which the target gene is being expressed.
  • the promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed “control sequences") is necessary to express a given gene.
  • control sequences also termed “control sequences”
  • the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • in-vitro generally refers to a biological reaction which occurs in an artificial environment outside a living organism, which is usually conducted in a laboratory using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis to be performed.
  • % homology is used interchangeably herein with the term “% identity” herein and normally refers to the level of nucleic acid or amino acid sequence identity between the nucleic acid sequence that encodes any one of the inventive polypeptides or the inventive polypeptide's amino acid sequence, when aligned using a sequence alignment program.
  • 80% homology means the same thing as 80% sequence identity determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence identity over a length of the given sequence.
  • Exemplary levels of sequence identity include, but are not limited to, 80, 85, 90, 95, 98% or more sequence identity to a given sequence, e.g., the coding sequence for any one of the inventive polypeptides, as described herein.
  • Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly accessible at www.ncbi.nlm.nih.gov/BLAST.
  • BLAST programs e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly accessible at www.ncbi.nlm.nih.gov/BLAST.
  • Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases.
  • the BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases.
  • a preferred alignment of selected sequences in order to determine "% identity" between two or more sequences is performed using for example, the CLUSTAL-W program.
  • primer is an oligonucleotide capable of binding to a target nucleic acid sequence and priming the nucleic acid synthesis.
  • An amplification oligonucleotide as defined herein will preferably be 10 to 50, most preferably 15 to 25 nucletides in length.
  • the amplification oligonucleotides of the present invention may be chemically synthesized.
  • nucleic acids comprising nucleotide sequences are the conventional one-letter abbreviations.
  • the naturally occurring encoding nucleotides are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • nucleic acid sequences presented herein is the 5' ⁇ 3'direction.
  • the term "complementary" and derivatives thereof are used in reference to pairing of nucleic acids by the well-known rules that A pairs with T or U and C pairs with G. Complement can be "partial” or “complete”. In partial complement, only some of the nucleic acid bases are matched according to the base pairing rules; while in complete or total complement, all the bases are matched according to the pairing rule. The degree of complement between the nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands as well known in the art. This may be of particular in detection method that depends upon binding between nucleic acids.
  • DNA sequences of the invention were generated by sequencing reactions and may contain minor errors which may exist as misidentified nucleotides, insertions, and/or deletions. However, such minor errors, if present, should not disturb the identification of the sequences as a gene of M. phaseolina that encodes an enzyme of industrial interest, and are specifically included within the scope of the invention.
  • SEQ ID No. 2 is provided which identifies a nucleotide sequence of the opening reading frame (ORF) of an identified gene.
  • the genomic sequences of the gene identified by SEQ ID No. 1 is provided.
  • SEQ ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, and 62, and/or any mixtures/combinations thereof are provided each of which identifies a nucleotide sequence of the opening reading frame (ORF) of an identified gene.
  • the disclosure also provides the genomic sequences of the genes identified by SEQ ID Nos. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 55, 58, and 61, and/or any mixtures/combinations thereof, are provided.
  • gene refers to (i) a gene comprising and/or consisting at least one of the nucleotide sequences and/or fragments thereof that are set forth in SEQ ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61; (ii) any nucleotide sequence or fragment thereof that encodes the amino acid sequence that are set forth in SEQ ID Nos.
  • the polynucleotides that hybridize to the complements of the DNA sequences disclosed herein encode gene products, e.g., gene products that are functionally equivalent to a gene product encoded by one of the enzyme genes or fragments thereof.
  • gene sequences of the disclosure include not only degenerate nucleotide sequences that encode the amino acid sequences of SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63, but also degenerate nucleotide sequences that when translated in organisms other than M. phaseolina, would yield a polypeptide comprising one of the amino acid sequences of SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63, or a fragment thereof.
  • the nucleotide sequences of the invention can be used as genetic markers and/or sequence markers to aid the development of a genetic, physical, or sequence map of the M. phaseolina genome.
  • the nucleotide sequences and corresponding gene products of the invention can also be used to detect the presence of M. phaseolina.
  • Hybridization and antibody-based methods well known in the art can be used to determine the presence and concentration of the nucleotide sequences and corresponding gene products of the invention.
  • nucleotide sequences can also be used for identifying inhibitors of the enzymes which may have therapeutic effects, given the fact that the enzymes may play a role in the invasion of a host during an infection.
  • homologs or orthologs of the genes of the invention which can be present in M. phaseolina and other fungal species are also included. Particularly preferred are homologs or orthologs in filamentous fungi. These enzyme genes can be identified and isolated by molecular biological techniques well known in the art.
  • Fungi includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota ( Hawksworth DL, Kirk PM, Sutton BC, Pegler DN. Ainsworth and Bisby's Dictionary of the Fungi (8th Ed.). 1995; CAB International, Wallingford, United Kingdom. 616p ) and yeast.
  • Representative groups of Ascomycota include, e.g., Neurospora, Penicillium, Aspergillus.
  • Representative groups of Basidiomycota include mushrooms, rusts, and smuts.
  • Representative groups of Chytridiomycota include Allomyces, Blastocladiella, Coelomomyces.
  • Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.
  • filamentous fungi include all filamentous forms of fungi.
  • the filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is done by hyphal elongation and carbon catabolism is obligately aerobic.
  • the present invention also provides fungal nucleotide sequences that are hybridizable to the polynucleotides of the genes.
  • the disclosure includes an isolated nucleic acid comprising and/or consisting of a nucleotide sequence that is at least 50% identical to a nucleotide sequence selected from the group comprising and/or consisting of: SEQ ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61, and/or any mixtures/combinations thereof.
  • the disclosure also includes an isolated nucleic acid comprising a fungal nucleotide sequence that hybridizes under medium stringency conditions to a second nucleic acid that consists and/or comprises of a nucleotide sequence selected from the group comprising and/or consisting of SEQ ID No. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61, and/or any mixtures/combinations thereof.
  • the disclosure also includes an isolated nucleic acid comprising a fungal nucleotide sequence that encodes a polypeptide the amino acid sequence of which is at least 50% identical to an amino acid sequence selected from the group comprising and/or consisting of SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63.
  • nucleotide sequences in accordance with the disclosure still further include fungal nucleotide sequences which are at least 40% identical to the nucleotide sequences set forth in SEQ ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61.
  • nucleic acid probes preferably detectably labeled, consisting of any one of the nucleotide sequences of SEQ ID No. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61 are included in the disclosure.
  • Hybridization conditions can be preferably of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived.
  • cDNA screening can also identify clones derived from alternatively spliced transcripts in the same species.
  • the labeled probe can be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • Low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. (Details in Sambrook J, Russell DW.
  • a homologous gene sequence can be isolated by performing a polymerase chain reaction (PCR) using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the gene of interest.
  • the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from the organism of interest.
  • the PCR product can be sub-cloned and sequenced to ensure that the amplified sequences represent the sequences of a homologous enzyme gene sequence.
  • the PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods well known to those of ordinary skill in the art.
  • the labeled fragment can be used to screen a genomic library.
  • the M. phaseolina gene sequences can be used in developing modified or novel enzymes that exhibit particularly desirable chemical and/or physical characteristics. Because of the apparent relatedness/similarity of the amino acid sequences among the enzymes of M. phaseolina and other filamentous fungi, the structure of an enzyme of another fungus can be used to predict the structure of the M. phaseolina enzyme, and aid in the rational modification of the M. phaseolina enzyme for useful and superior properties.
  • the sequences provided can also be used as preparatory materials for the rational modification or design of novel enzymes with characteristics that enable the enzymes to perform better in demanding processes.
  • the gene nucleotide sequences can be altered by random and site-directed mutagenesis techniques or directed molecular evolution techniques, such as but not limited to the methods described in ( Arnold FH. Protein engineering for unusual environments. Curr. Opinion Biotechnol. 1993;4:450-455 ), oligonucleotide-directed mutagenesis ( Reidhaar-Olson JF, Sauer RT. Combinatorial cassette mutagenesis as a probe of the informational content of protein sequences. Science, 1988; 241:53-57 ), chemical mutagenesis ( Eckert KA, Drinkwater NR.
  • the 747 bp long polynucleotide illustrated in SEQ ID No. 2 is the full length cDNA clone encoded pectate lyase protein exhibiting an open reading frame encoding 248 amino acid polypeptide, as in SEQ ID No. 3, with a calculated molecular mass about 26 kD.
  • SEQ ID No. 2 Through SMART analysis of SEQ ID No. 2, it reveals presence of Pfam pectate lyase domain in the sequence.
  • Pectate lyase is an extracellular enzyme that catalyzes the eliminative cleavage of pectate to produce oligosaccharides with 4-deoxy-alpha-D-gluc-4-enuronosyl groups at their non-reducing ends. This enzyme is involved in the degradation of pectin. is
  • the disclosure also relates to (a) nucleic acid vectors that comprise and/or consist a nucleotide sequence comprising any of the foregoing sequences of the genes and/or their complements; (b) expression constructs that comprise a nucleotide sequence consisting and/or comprising any of the foregoing coding sequences of the genes operably linked with a regulatory element that directs the expression of the coding sequences; and (c) recombinant host cells that comprise and/or consist any of the foregoing sequences of the gene, including coding regions operably linked with a regulatory element that directs the expression of the coding sequences in the host cells.
  • the techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.
  • the various sequences may be joined in accordance with known techniques, such as restriction, joining complementary restriction sites and ligating, blunt ending by filling in overhangs and blunt ligation or the like.
  • Poly linkers and adapters may be employed, when appropriate, and introduced or removed by known techniques to allow for ease of assembly of the DNA vectors and expression constructs.
  • a large number of vectors are available for cloning and genetic manipulation. Normally, cloning can be performed in E. coli.
  • vectors that comprise an enzyme gene sequence of the invention may further comprise replication functions that enable the transfer, maintenance and propagation of the vectors in one or more species of host cells, including but not limited to E. coli cells, filamentous fungal cells, yeast cells, and Bacillus cells.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • Expression construct of the invention comprises and/or consists of a promoter, a nucleotide sequence encoding for a gene sequence of the invention, a transcription termination sequence and selectable marker (optional). Any method known in the art for introducing this expression construct into a host cell can be used. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton MM, Hames JE, Timberlake WE. Transformation of Aspergillus nidulans by using a trpC plasmid. PNAS,1984; 81(5):1470-1474 .
  • Suitable methods for transforming Fusarium species are described by Malardier L, Daboussi MJ, Julien J, Roussel F, Scazzocchio C, Brygoo Y. Cloning of the nitrate reductase gene (niaD) of Aspergillus nidulans and its use for transformation of Fusarium oxysporum. Gene 1989; 78:147-156 , and WO 96/00787 . Yeast may be transformed using the procedures described by Becker DM, Guarente L. High-efficiency transformation of yeast by electroporation.
  • the enzymes of the present invention are produced by a fungal cell.
  • the expression host cell is a filamentous fungal cell which has been used in large scale industrial fermentation.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • an expression host is selected which is capable of the efficient secretion of their endogenous proteins.
  • a host cell may also be chosen for deficiencies in extracellular protease activities since the secreted enzyme may be degraded in the culture medium.
  • the present invention also relates to methods for producing a polypeptide of the present invention, comprising: (i) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (ii) recovering the polypeptide.
  • the cell is M. phaseolina.
  • the disclosure also relates to methods for producing a polypeptide, comprising: (i) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a nucleotide sequence of SEQ ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 or 61, wherein the nucleotide sequence encodes a polypeptide which comprises or consists of the mature polypeptide of SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 or 63, and (ii) recovering the polypeptide.
  • the expression host cells or transformants may be cultivated in a suitable nutrient medium for growth and expression of proteins using methods well known in the art.
  • the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art (see In: More Gene Manipulations in Fungi. Bennett J W, Lasure L., (eds). 1991; Academic Press, San Diego, CA ). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.
  • the polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. An enzyme assay may be used to determine the activity of the polypeptide.
  • the produced polypeptide may be recovered using methods known in the art.
  • the polypeptide may be recovered in various methods from the nutrient medium by conventional procedures including, but not limited to, filtration, centrifugation, extraction, spray-drying, evaporation and precipitation or combination thereof.
  • polypeptides of the present invention and other polypeptides of the disclosure may be purified by a variety of procedures that are well known in the art including, but not limited to, chromatography method (such as ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (such as isoelectric focusing), differential solubility (such as ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure polypeptides (see details in Protein Purification, Principles, High Resolution Methods and Applications. Janson JC, Rydén L. (eds(). 1989; VCH Publishers Inc., New York ).
  • the disclosure also relates those gene products (e.g., RNA or proteins) that are encoded by the gene sequences set forth in SEQ ID Nos. 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59 and 62.
  • the enzyme gene products also includes those RNA or proteins that are encoded by the genomic sequences of the genes as set forth in SEQ ID Nos. 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58 and 61.
  • the enzymes comprise an amino acid sequence selected from the group comprising and/or consisting of SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63.
  • the enzymes display at least one of the activities of an enzyme selected from the group comprising and/or consisting of pectate lyase, pectate lyase C, pectinesterase and rhamnogalacturonase.
  • the enzyme gene products can be readily produced, e.g., by synthetic techniques or by methods of recombinant DNA technology using techniques that are well known in the art (See, Creighton TE. Proteins: Structures and Molecular Principles, 1983; W. H. Freeman and Co., N.Y .)
  • the methods and compositions of the disclosure also include proteins and polypeptides that represent functionally equivalent gene products.
  • Such functionally equivalent gene products include, but are not limited to, natural variants of the polypeptides having an amino acid sequence set forth in SEQ ID Nos. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60 and 63.
  • Such equivalent gene products can contain, e.g. deletions, additions or substitutions of amino acid residues within the amino acid sequences encoded by the enzyme gene sequences described above, but which result in a silent change, thus producing a functionally equivalent product.
  • Amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved.
  • Another embodiment of the present invention further includes enzymes of the present invention in solid form.
  • Enzymes in solid form or enzyme granulate can be used, for example, in solid detergent and in animal feed.
  • Methods of making solid forms of enzymes are well known in the art, such as but not limited to prilling (spray-cooling in a waxy material), extrusion, agglomeration, or granulation (dilution with an inert material and binders).
  • Solid enzymatic compositions comprising a solid form of an enzyme of the invention, in the form of mixed powder, tablets, and the like, is contemplated.
  • Genomic DNA was isolated from M. phaseolina strain ms6 using the procedures described by Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces Genetics, 2000; John Innes Foundation, Norwich, UK, pp. 162-208 . Briefly, mycelia were scraped from a stock Petri plate using inoculating loop and inoculated into a liquid potato-dextrose media in a conical flask. The conical flask was incubated without shaking for about 72 hours at 30°C. The mycelia grow on the surface at the air-medium interface.
  • the mycelia were harvested using a sterile toothpick and placed between sterile paper towels and several were washed with physiological solution (Sodium phosphate buffer pH 7.0). The mycelia were squeezed to remove excess liquid and the harvested mycelia were allowed to air dry for 30 minutes. The semi-dry mycelia were placed in liquid nitrogen and ground to generate fine powder and finally isolate the DNA following the protocol describe in Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces Genetics, 2000; John Innes Foundation, Norwich, UK, pp. 162-208 .
  • the primers used in the study were either designed from the manually curated transcriptome and the "gene models" predicted from the genomic sequences of M. phaseolina ms6, by choosing the sequences manually with complete ORFs or using databases where similar genes have been successfully isolated from other plants. Comparative bioinformatic analysis of the nucleotide sequences obtained from transcriptome were carried out using NCBI BLAST, BLASTP, RPS-BLAST, BLASTX and PSI-BLAST to identify homologues of the related genes and for the proper identification of gene. Nucleotide sequence alignments were performed using clustalW version 1.82 whenever multiple sequences were found from the "gene pool”. The alignment was then edited. Gene specific primers (both forward and reverse) were selected manually or through Primer 3 plus tool and the primers were custom synthesized.
  • oligonucleotides used in this study were synthesized and HPLC purified by the supplier and procured from Integrated DNA Technologies (IDT). Stock solution of 100 pmol were prepared in autoclaved ddH 2 O and stored at -20°C, in aliquots for use. Oligonucleotides Sequences used as primers for PCR Gene name SEQ ID No.
  • Pectate lyase 1 Forward TCATCACCGACCCACAATCGC (SEQ ID NO: 64) 859 Reverse CTTAGCAAGCGGGAGCCGTC (SEQ ID NO: 65) Pectate lyase 4 Forward CAGACAACGGCGTCGACAGT (SEQ ID NO: 66) 1029 Reverse ACGGACCACATCCAACGCGA (SEQ ID NO: 67) Pectate lyase 7 Forward ATGCCCTGGTACCCTGCCCC (SEQ ID NO: 68) 1010 Reverse GAAAGGTCCCGGGCTCACGC (SEQ ID NO: 69) Pectate lyase 10 Forward CCGCTCCTCTGTGCGTTGTTGAA (SEQ ID NO: 70) 1036 Reverse ACAGCGATAGAGCCGCAACACG (SEQ ID NO: 71) Pectate lyase 13 Forward TTTCCTCACCACCACTTCCCCTCT (SEQ ID NO: 72) 982 Reverse CG
  • the PCR reaction (50 ⁇ L) contained 1 ⁇ L of cDNA, 20 pmoles of each primers, 5 ⁇ L of 10X PCR Buffer, 5 ⁇ L of 2.5 mM dNTP mix and 1.0 unit of PfuTaq DNA polymerase.
  • PCR was carried out in Thermal Cycler (Applied Biosystems) using the following conditions: initial denaturation for 5 minutes ("min") at 95°C followed by 35 cycles of denaturation at 95°C for 30 seconds (“sec”), annealing at 59-61°C for 30 sec and extension at 72°C for 1 to 2.0 min depending on the length of the targeted gene, with a final extension at 72°C for 7 min.
  • the PCR product was analyzed by 1% agarose gel using 1XTAE buffer and the amplicon was eluted from the gel using QIAGEN gel extraction kit following the manufacturer's instructions.
  • the purified PCR product was ligated into pCR®8/GW/TOPO® TA cloning kit (Invitrogen) and transformed into competent E. coli cells (Invitrogen). Plasmids were isolated from putative colonies using QIAprip Spin Miniprep Kit (QIAGEN) following the manufacturer's instructions. The presence of the insert was checked by using the gene specific primers and positive plasmids were subjected to Sequencing.
  • the nucleotide sequence and the amino acid sequence were analyzed by BLASTN and BLASTP programs respectively.
  • the sequences reported from other plants were aligned with ClustalW.
  • Phylogenetic analysis was carried out using the Neighbour Joining (NJ).

Claims (15)

  1. Isolierte Polynukleotidsequenz, umfassend eine Nukleotidsequenz, welche mindestens zu 80 % mit der in SEQ ID NO 1 oder 2 dargelegten Nukleotidsequenz identisch oder dem Komplement davon ist, wobei das Polynukleotid für eine Pektatlyase kodiert.
  2. Isolierte Nukleinsäuresonde, die unter mittleren Stringenzbedingungen zu einer isolierten Polynukleotidsequenz hybridisiert, umfassend eine in SEQ ID No 1 oder 2 dargelegte Nukleotidsequenz oder das Komplement davon, wobei das Polynukleotid für eine Pektatlyase kodiert, und wobei die mittleren Stringenzbedingungen die Hybridisierung zu Filter-gebundener DNA in 6x Natriumchlorid/Natriumcitrat (SSC) bei ca. 45 °C, gefolgt von einer oder mehreren Wäschen in 0,2xSSC/0,1 % SDS bei ca. 50 bis ca. 65 °C einschließt.
  3. Isolierte Polynukleotidsequenz nach Anspruch 1 oder isolierte Nukleinsäuresonde nach Anspruch 2, welche genomische DNA ist.
  4. Isolierte Polynukleotidsequenz nach Anspruch 1 oder isolierte Nukleinsäuresonde nach Anspruch 2, welche cDNA ist.
  5. Isolierte Polynukleotidsequenz nach Anspruch 1 oder isolierte Nukleinsäuresonde nach Anspruch 2, welche RNA ist.
  6. Isolierte Polynukleotidsequenz nach Anspruch 1 oder isolierte Nukleinsäuresonde nach Anspruch 2, welche einzelsträngig ist.
  7. Nukleinsäuremolekül, umfassend die isolierte, nicht innerhalb einer Kodiersequenz durch Stopcodon unterbrochene Polynukleotidsequenz nach Anspruch 1, die für ein heterologes Protein oder Peptid kodiert.
  8. Rekombinanter Vektor, umfassend die isolierte Polynukleotidsequenz nach Anspruch 1.
  9. Expressionskonstrukt, umfassend die isolierte Polynukleotidsequenz nach Anspruch 1, wobei die Polynukleotidsequenz funktionsmäßig mit einer regulatorischen Nukleotidsequenz verbunden ist, enthaltend transkriptionelle oder translationale regulatorische Signale oder beide, die Ausdruck der Nukleotidsequenz in einer Wirtszelle steuert.
  10. Gentechnisch gefertigte Wirtszelle, umfassend die isolierte Polynukleotidsequenz nach Anspruch 1.
  11. Verfahren zum Herstellen eines Polypeptids, umfassend die folgenden Schritte:
    i. Kultivieren einer Zelle, die mit einem Expressionskonstrukt nach Anspruch 9 unter wirksamen Bedingungen umgewandelt ist, umdas Polypeptid herzustellen, und
    ii. Isolieren des Polypeptids.
  12. Isoliertes Polypeptid, umfassend eine Aminosäuresequenz, welche mindestens zu 80 % mit einer in SEQ ID NO 3 Aminosäuresequenz identisch ist und die katalytische Aktivität von Pektatlyase zeigt.
  13. Chimäres Protein, umfassend ein Polypeptid nach Anspruch 12, kondensiert über eine kovalente Bindung an eine Aminosäuresequenz eines zweiten Polypeptids.
  14. Wirtszelle, umfassend das Expressionskonstrukt nach Anspruch 9.
  15. Transgene Pilze von M. phaseolina, umfassend das Expressionskonstrukt nach Anspruch 9.
EP13829902.9A 2012-08-16 2013-08-15 Pektinabbauende enzyme aus macrophomina phaseolina und verwendungen davon Active EP2885407B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PL13829902T PL2885407T3 (pl) 2012-08-16 2013-08-15 Enzymy rozkładające pektynę z macrophomina phaseolina oraz ich zastosowania

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201261683914P 2012-08-16 2012-08-16
PCT/US2013/055198 WO2014028772A2 (en) 2012-08-16 2013-08-15 Pectin degrading enzymes from macrophomina phaseolina and uses thereof

Publications (3)

Publication Number Publication Date
EP2885407A2 EP2885407A2 (de) 2015-06-24
EP2885407A4 EP2885407A4 (de) 2016-05-11
EP2885407B1 true EP2885407B1 (de) 2018-06-13

Family

ID=50101609

Family Applications (1)

Application Number Title Priority Date Filing Date
EP13829902.9A Active EP2885407B1 (de) 2012-08-16 2013-08-15 Pektinabbauende enzyme aus macrophomina phaseolina und verwendungen davon

Country Status (16)

Country Link
US (1) US9765322B2 (de)
EP (1) EP2885407B1 (de)
JP (1) JP6644547B2 (de)
KR (1) KR102043363B1 (de)
CN (1) CN104837991A (de)
AU (1) AU2013302535B2 (de)
DK (1) DK2885407T3 (de)
ES (1) ES2674922T3 (de)
HK (1) HK1213597A1 (de)
IN (1) IN2015DN02049A (de)
MY (1) MY169809A (de)
PL (1) PL2885407T3 (de)
PT (1) PT2885407T (de)
TR (1) TR201809053T4 (de)
WO (1) WO2014028772A2 (de)
ZA (1) ZA201501635B (de)

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK122686D0 (da) 1986-03-17 1986-03-17 Novo Industri As Fremstilling af proteiner
GB8702475D0 (en) 1987-02-04 1987-03-11 Ciba Geigy Ag Expression system
ES2063162T5 (es) 1988-07-28 2002-11-16 Novartis Ag Nuevo sistema de expresion.
DE3908813A1 (de) 1989-03-17 1990-09-20 Roehm Gmbh Verfahren zur expression eines aus aspergillus niger stammenden gens in einem aspergillus
EP0675950A1 (de) 1992-12-23 1995-10-11 Novo Nordisk A/S Ein enzym mit polygalakturonaseaktivität
US6117679A (en) 1994-02-17 2000-09-12 Maxygen, Inc. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
CN1151762A (zh) 1994-06-30 1997-06-11 诺沃诺尔迪斯克生物技术有限公司 非毒性、非产毒性、非致病性镰孢属表达系统及所用启动子和终止子
CN1182451A (zh) * 1995-03-17 1998-05-20 诺沃挪第克公司 新的内切葡聚糖酶
US5939250A (en) 1995-12-07 1999-08-17 Diversa Corporation Production of enzymes having desired activities by mutagenesis
US5965408A (en) 1996-07-09 1999-10-12 Diversa Corporation Method of DNA reassembly by interrupting synthesis
US6171820B1 (en) 1995-12-07 2001-01-09 Diversa Corporation Saturation mutagenesis in directed evolution
JP3534607B2 (ja) 1997-04-09 2004-06-07 花王株式会社 洗浄剤組成物
US7214786B2 (en) * 2000-12-14 2007-05-08 Kovalic David K Nucleic acid molecules and other molecules associated with plants and uses thereof for plant improvement
CN100482791C (zh) * 2002-05-14 2009-04-29 诺和酶股份有限公司 果胶酸裂解酶变体
EP1507857A1 (de) 2002-05-30 2005-02-23 DSM IP Assets B.V. Neue pektinasen und deren verwendungen
CN102016041A (zh) * 2008-02-29 2011-04-13 中央佛罗里达大学研究基金会有限公司 植物降解材料的合成和应用
WO2010070146A1 (en) * 2008-12-19 2010-06-24 Danisco A/S Process for production of an enzyme product
WO2010101158A1 (ja) * 2009-03-02 2010-09-10 住友商事株式会社 クロストリジウム セルロボランス由来新規遺伝子及びその利用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
IN2015DN02049A (de) 2015-08-14
ZA201501635B (en) 2016-01-27
MY169809A (en) 2019-05-16
EP2885407A2 (de) 2015-06-24
WO2014028772A2 (en) 2014-02-20
ES2674922T3 (es) 2018-07-05
KR20150042849A (ko) 2015-04-21
US20150259667A1 (en) 2015-09-17
KR102043363B1 (ko) 2019-11-11
CN104837991A (zh) 2015-08-12
PL2885407T3 (pl) 2018-09-28
AU2013302535A1 (en) 2015-03-26
JP2015529071A (ja) 2015-10-05
WO2014028772A3 (en) 2014-05-08
US9765322B2 (en) 2017-09-19
EP2885407A4 (de) 2016-05-11
TR201809053T4 (tr) 2018-07-23
JP6644547B2 (ja) 2020-02-12
HK1213597A1 (zh) 2016-07-08
DK2885407T3 (en) 2018-07-16
AU2013302535B2 (en) 2018-12-06
PT2885407T (pt) 2018-07-02
BR112015003086A2 (pt) 2018-06-26

Similar Documents

Publication Publication Date Title
KR102647766B1 (ko) 클래스 ii, 타입 v crispr 시스템
US11624061B2 (en) Compositions and methods for enzyme immobilization
Silva et al. Identification, expression, and characterization of a novel bacterial RGI Lyase enzyme for the production of bio-functional fibers
CN112074530A (zh) 包含粘度降低表型的丝状真菌菌株
EP2885407B1 (de) Pektinabbauende enzyme aus macrophomina phaseolina und verwendungen davon
EP1200574A1 (de) Cellobiohydrolase reduzierte glykolysierungsvarianten: cbhin45a; cbhin270a und cbhin384a
JP2004313074A (ja) 新規α−１，２−マンノシダーゼおよびその遺伝子、ならびに該酵素を用いたα−マンノシル糖化合物の製造方法
KR102358538B1 (ko) 유전자 총법을 이용한 미세조류의 교정 방법
EP2885406B1 (de) Cellulose- und/oder hemicelluloseabbauende enzyme aus macrophomina phaseolina und verwendungen davon
BR112015003086B1 (pt) Sequência de polinucleotídeo recombinante, vetor de expressão recombinante e célula hospedeira procariótica geneticamente engenheirada
CN114854717B (zh) 一种脂肪酶及其编码基因与应用
US11976266B2 (en) Filamentous fungal strains comprising reduced viscosity phenotypes
JP2014168417A (ja) β−キシロシダーゼ、及びβ−キシロシダーゼをコードする遺伝子
CA3225082A1 (en) Enzymes with ruvc domains
BR112015003085B1 (pt) Molécula de ácido nucleico recombinante, constructo recombinante de expressão e célula hospedeira procariótica engenheirada geneticamente
CN115838705A (zh) 一种脂肪酶突变体
CN117821422A (zh) 核酸内切酶iv突变体及其应用

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20150306

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAX Request for extension of the european patent (deleted)
RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 9/88 20060101AFI20151207BHEP

A4 Supplementary search report drawn up and despatched

Effective date: 20160407

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 9/88 20060101AFI20160401BHEP

17Q First examination report despatched

Effective date: 20161207

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20180223

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1008538

Country of ref document: AT

Kind code of ref document: T

Effective date: 20180615

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: RO

Ref legal event code: EPE

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Ref document number: 2885407

Country of ref document: PT

Date of ref document: 20180702

Kind code of ref document: T

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20180626

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2674922

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20180705

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602013039008

Country of ref document: DE

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: TR-IP CONSULTING LLC, CH

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20180711

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 6

REG Reference to a national code

Ref country code: NO

Ref legal event code: T2

Effective date: 20180613

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

REG Reference to a national code

Ref country code: GR

Ref legal event code: EP

Ref document number: 20180401839

Country of ref document: GR

Effective date: 20190109

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181013

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602013039008

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20190314

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20180613

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180815

REG Reference to a national code

Ref country code: CH

Ref legal event code: PCAR

Free format text: NEW ADDRESS: ROUTE DU COUTSET 18, 1485 NUVILLY (CH)

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20180613

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 1008538

Country of ref document: AT

Kind code of ref document: T

Effective date: 20180613

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230526

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: NL

Payment date: 20230826

Year of fee payment: 11

Ref country code: LU

Payment date: 20230828

Year of fee payment: 11

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20230720

Year of fee payment: 11

Ref country code: RO

Payment date: 20230724

Year of fee payment: 11

Ref country code: NO

Payment date: 20230829

Year of fee payment: 11

Ref country code: IT

Payment date: 20230822

Year of fee payment: 11

Ref country code: IE

Payment date: 20230828

Year of fee payment: 11

Ref country code: GB

Payment date: 20230828

Year of fee payment: 11

Ref country code: ES

Payment date: 20230901

Year of fee payment: 11

Ref country code: CZ

Payment date: 20230721

Year of fee payment: 11

Ref country code: CH

Payment date: 20230903

Year of fee payment: 11

Ref country code: BG

Payment date: 20230810

Year of fee payment: 11

Ref country code: AT

Payment date: 20230719

Year of fee payment: 11

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20230827

Year of fee payment: 11

Ref country code: PT

Payment date: 20230720

Year of fee payment: 11

Ref country code: PL

Payment date: 20230725

Year of fee payment: 11

Ref country code: HU

Payment date: 20230725

Year of fee payment: 11

Ref country code: GR

Payment date: 20230829

Year of fee payment: 11

Ref country code: FR

Payment date: 20230825

Year of fee payment: 11

Ref country code: DK

Payment date: 20230829

Year of fee payment: 11

Ref country code: DE

Payment date: 20230829

Year of fee payment: 11

Ref country code: BE

Payment date: 20230828

Year of fee payment: 11